Restricted access media (RAM) have been used for many years in work with biological samples to combine the features of size exclusion chromatography with partition or ion-exchange chromatography. The support in RAM is typically porous silica or a porous polymer having a non-adsorptive and hydrophilic outer surface and an interior stationary phase that can retain analytes through partitioning or ionic interactions. This provides a medium in which only low mass substances are retained, while larger agents such as proteins elute in the excluded volume. RAM have been used for analysis of drugs, peptides, and endogenous substances in complex samples such as serum, blood, urine, and cell cultures.
Many small molecules such as drugs and hormones bind proteins and/or other agents that are present in a sample. This gives rise to both a free (non-bound) form and a bound form of the small molecule in the sample. Traditional methods for separating free and bound forms of small molecules include equilibrium dialysis and ultrafiltration, which can involve long analysis times, are often labor intensive, and usually require additional analytical methods for actual measurement of the free form. These methods involve a size separation between the free form and bound form. Non-affinity restricted access media columns have been used for separation of free and bound forms, however, these columns have given only partial separation and work for only some analytes. Ultrafast extraction of free forms by small immobilized antibody columns have been reported, however, these assays require using an antibody support that can recognize and bind the free form without having significant interactions with the bound form. Accordingly, a need exists for a media having high selectivity towards free forms of the small molecule but without significant interactions with bound forms and methods for making these media.
One challenge presented by immobilized or covalently linked molecules is to attach them to supports while retaining their behavior in their native form. Covalently linking or immobilization of molecules directly to the support may affect the molecule's activity if the molecule is linked at or near its active site. For example, antibodies attached to a support by their Fab fragment may decrease and/or lose their ability to bind antigen. Covalent immobilization may also result in multisite attachment and random orientation of the molecule, which often leads to decreased or complete loss of activity. Noncovalent immobilization techniques can involve the adsorption of a ligand binding agent to a surface, the binding of one ligand binding agent to a second ligand binding agent, or the formation of a complex between the ligand binding agent and support. Entrapment or encapsulation techniques involve the physical containment of a molecule in a support. Previously described entrapment methods are limited to particular types of supports (i.e., sol gels) and are useful only with relatively small volume systems (i.e., capillary columns in HPLC). Accordingly, a need exists for noncovalent retention of molecules that is applicable to a variety of different support materials and molecules to produce supports containing noncovalently linked agents that closely mimic the behavior of these same agents in their native unbound form.